An ideal vaccine for human immunodeficiency virus type-1 (HIV-1) should have the capacity to stimulate strong, broadly reactive humoral and cellular immunity, especially CD8+ T cell responses. Measurement of these responses in clinical trials of candidate vaccines is of the utmost importance, and is dependent entirely upon in vitro methodologies. Optimal assays should be sensitive, specific, easy to perform and highly reproducible. We have developed an intracellular cytokine-staining (ICS) assay for quantifying CD8+ T cells specific to entire HIV viral proteins. We hypothesize that this new assay has the potential to satisfy the requirements for an optimal assay, and that it would be able to detect low level responses that were not appreciated with older methodologies, such as the limiting dilution analysis (LDA). To test this hypothesis, we will further characterize and standardized our current ICS format. Peripheral blood mononuclear cells (PBMCs) obtained from HIV-uninfected individuals and from HIV-infected patients who are long term non-progressors (LTNPs), as well as cloned T cell lines with known HIV-specificity, will be utilized to determine the sensitivity, specificity and reproducibility of the ICS assay. In addition, we will validate the new ICS assay by comparing it with the other two newly developed quantitative assays, i.e. the major histocompatibility complex (MHC) class I tetramer assay, and the enzyme-linked immunospot (ELISPOT) assay in detecting CD8+ T cell responses using PBMC from the same individuals. Subsequently, we will optimize the ICS assay to measure CD8+ T cell responses among patients attending the University of Rochester (UR) HIV Clinic. Finally, we will revalidate the ICS assay in PBMC obtained from the HIV Vaccine Trials Network to assess levels of CD8+ T cell responses among recipients of HIV vaccines.